Throughout this application, various publications are referenced within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Presently, there are no commercially effective tests to provide a rapid diagnostic approach to individuals with adrenocortical disorders of metabolism and development such as the X-linked cytomegalic form of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to mutations in DAX-1, a new member of the nuclear hormone receptor gene superfamily. Left untreated, the results of these disorders include failure to achieve pubertal growth, sexual maturity, and eventually death.
AHC is an inherited disorder of adrenal gland development. The disorder results in adrenal insufficiency early in infancy, with low serum concentration of glucocorticoids, mineralocorticoids and androgens, and failure to respond to ACTH stimulation. AHC is a rare disorder with an overall estimated frequency of 1:12,500 live births.
Clinical signs and symptoms of infants with adrenal hypoplasia include poor feeding, failure to gain weight, hyperpigmentation, vomiting, diarrhea, vascular collapse, and sudden death. Dehydration, hyponatremia, hyperkalemia, acidosis, and hypoglycemia are common biochemical findings characteristic of combined glucocorticoid and mineralocorticoid deficiency.
HH is commonly associated with the X-linked form of the disease, and is generally noted at the expected time of puberty. It is not clear if this form of HH is of pituitary or hypothalamic origin. HH often is noted in boys with X-linked AHC at the expected time of pubertal maturation. Furthermore, abnormalities of the genitourinary system appear to occur with increased frequency in boys with AHC, and these include: cryptorchidism, hypospadias, small external genitalia, ureteral reflux, and urethral stenosis.
It would be of significant value to society to be able to use information to develop tests to determine the genetic imprint of AHC or HH.
There is a need for laboratory tests that identify those having the propensity to develop AHC and HH. There is currently a need to develop (1) methods to identify patients who need treatment along with the appropriate type of treatment, (2) apparatus, and (3) systems to determine the characteristics of AHC and HH.
Members of the nuclear hormone receptor superfamily are ligand dependent transcription factors which modulate a large number of essential cellular processes. Members of the superfamily regulate a myriad of pathways in higher organisms ranging from development and morphogenesis to reproduction, behavior and homeostasis. The superfamily consists of receptors for steroid hormones (e.g. corticosteroids, estrogens, progestins, and androgens), steroid derivatives (dihydroxy vitamin D3) and non-steroids (thyroid hormone and retinoids). In addition, there are members of this superfamily for which a ligand has not yet been identified, the so-called xe2x80x9corphan receptors,xe2x80x9d or members which have lost ligand binding function (e.g., thyroid hormone receptor xcex12; TRxcex12).
Nuclear hormone receptors are localized within a cell and, in contrast to receptors for peptide hormones, are not cell-membrane associated. They mediate their function in the cell nucleus by directly influencing gene expression. The nuclear hormone receptors have high affinity for their ligand which is in the range of 10-9 to 10-11 M. It is believed that the lipophilic hormone passes through the cell membrane without the help of specialized membrane bound accessory proteins and binds to the receptor within the cell. Hormone binding has a drastic effect in transforming the receptor by changing its conformation and thereby altering its function and activity.
Hormones which act by way of nuclear hormone receptors affect numerous tissues and pathways. One such group of hormones is the corticosteroids, which can be divided into glucocorticoids (cortisol and corticosterone) and mineralocorticoids (aldosterone). They are synthesized in the adrenal cortex using cholesterol as a precursor. Also synthesized in the adrenal cortex are androgens, which affect growth, development of skeletal muscle and behavior.
The invention provides DAX-1 protein molecules. DAX-1 protein is a novel member of the nuclear hormone receptor superfamily and may bind a retinoic acid or other response element. DAX-1 is expressed in steroidogenic cells in mammals including the adrenal gland, ovaries, testes, hypothalamus and pituitary gland.
In one embodiment, the DAX-1 protein has the amino acid sequence beginning with methionine at position 1 and ending with isoleucine at position 470 as shown in FIG. 12.
Additionally, a DAX-1 protein molecule expressed by species other than the human species is encompassed within this invention. It will share substantial homology with the DAX-1 protein having the amino acid sequence beginning with methionine at position 1 and ending with isoleucine at position 470 as shown in FIG. 12. The data herein support the existence of the DAX-1 protein expressed by various species, e.g., from mammals to yeast (FIG. 10). A homologous sequence has been found also in C. elegans. 
The invention also provides nucleic acid molecules encoding the protein of the invention (FIG. 12). For example, in one embodiment the nucleic acid molecule is a DNA molecule. The genomic DNA molecule includes both the intron and exons of the sequence which encodes DAX-1. Alternatively, the nucleic acid molecule is a cDNA molecule as shown in FIG. 12.
The proteins and nucleic acid molecules of the present invention are weapons that can be used as part of an arsenal of weapons against adrenocortical disorders of metabolism and development.
The invention also provides methods of gene therapy which utilize DAX-1 nucleic acid moleules. In one embodiment, a gene encoding a DAX-1 protein is transferred into steroidogenic cells. As a first step in this method, a vector comprising DNA encoding a DAX-1 protein is introduced into a producer cell which results in the integration of the vector into the cell. Producer cells into which the vector is integrated are then selected. As a final step, the producer cell is grafted proximate to the steroidogenic cells so that the steroidogenic cells are infected with the vector produced by the producer cell and the gene encoding DAX-1 is transferred to the steroidogenic cells. In a further embodiment, the vector used in the above method comprises a portion of the promoter region of the DAX-1 DNA sequence and a transgene. In a still further embodiment, the transgene is a therapeutic transgene.
The invention further provides a method for screening drugs which bind a DAX-1 protein. This method comprises, as a first step, transfecting a cell with (i) a vector capable of expressing at least the ligand binding domain of a DAX-1 gene linked with a DNA binding domain (DBD) sequence, and (ii) a construct capable of expressing a reporter gene and having a response element to which the DBD binds, wherein the construct is part of the vector or is separate therefrom. As a second step, the transfected cell is cultured under conditions permitting binding of the expressed DBD to the response element. Next, the transfected cell is contacted with a drug to be screened. The presence of a protein encoded by the reporter gene is then detected, the presence of the protein being indicative of the binding of the drug to DAX-1 protein.